Chronic stress is a well-known trigger for abdominal pain, a common yet poorly understood presentation in the clinical setting. We will examine the novel hypothesis that chronic stress activates epigenetic regulatory pathways resulting in enhanced abdominal pain (visceral hyperalgesia). Epigenetic pathways regulate gene expression by modifying the level of methylation of upstream promoter regions of genes (increased methylation decreases gene expression) and histone acetylation (increased histone acetylation enhances gene expression). Stress activates the hypothalamic- pituitary-adrenal axis resulting in increased blood levels of the hormone cortisol in humans and corticosterone (CORT) in rodents which regulate gene expression via the intracellular corticoid receptors GR and MR. Acute stress activates the anti-nociceptive endocannabinoid CB1 receptor gene expression which, in turn, inhibits the function of pro-nociceptive endovanilloid TRPV1 receptors. Chronic stress down-regulates CB1 expression and up-regulates TRPV1 expression. We propose that chronic stress will increase methylation of GR promoter sites resulting in decreased CB1 expression, and augment histone acetylation resulting in increased TRPV1 expression, culminating in visceral hyperalgesia. DNA methylation sites will be characterized using methylation-specific PCR analysis and pyrosequencing, and histone acetylation sites dissected using ChIP. We propose that chronic stress will modulate these pathways selectively in primary sensory dorsal root ganglion (DRG) neurons innervating the colon that transmit pain. Studies will be performed in DRGs obtained from chronic water-avoidance stressed rats, control rats treated with CORT in situ and control DRG explants treated with CORT in vitro. We will examine whether the formation of GR-MR and CB1-TRPV1 receptor complexes plays a role in stress-related visceral hyperalgesia in DRG neurons transfected with either dual labeled GR and MR, or CB1 and TRPV1 using FRET/TIRF microscopy. We hypothesize that visceral hyperalgesia will be linked to epigenetic mechanisms that selectively affect promoter sites for the GR, CB1 and TRPV1 receptors in nociceptive DRG neurons innervating the colon. This subpopulation will be identified using immunohistochemical markers and laser capture microscopy to harvest different subpopulations of DRG neurons in conjunction with quantitative PCR of relevant targets. Confirmation of the role of specific receptors (GR/MR) and signal transduction pathways (methylation/acetylation) to the changes observed in CB and TRP pathways will be confirmed using targeted delivery of specific agonist/antagonist drugs and gene silencing (si-RNA) reagents in situ, and correlation of these interventions with behavior, e.g. visceral motor response to colorectal distension. We believe that the proposed studies will have high impact regarding our understanding of how stress affects pain perception. Therapeutic interventions that target epigenetic mechanisms are touted as a pathway to personalized medicine in the future.